The determination of the presence or amount of certain constituents or analytes is useful in the diagnosis of disease and physical well-being. Compositions which behave similarly to how constituents present in human bodily fluids (e.g. blood, blood serum, plasma, spinal fluid or urine) behave are used in clinical laboratories. These compositions assist in the determination of whether the clinical instrumentation and procedures used by the laboratory to measure the constituents are accurate. These compositions are also used to calibrate the clinical devices which measure the amount or presence of the constituent in a sample. These compositions will be referred to hereinafter as control compositions or controls.
In addition, it is important that the analyte or analyte analog present in the control composition behave similarly to the corresponding analyte to be tested for in a patient's bodily fluid--that is, the control composition should mimic the patient sample.
Rapid and simple tests that can be used to accurately diagnose the occurrence of myocardial infarction ("MI") or distinguish other ischemic events such as unstable angina are extremely important. Cardiac troponin I (cTnI) and troponin T have recently become established as the markers of choice in evaluating cardiac distress. See, New England Journal of Medicine Volume 335 No. 18, pages 1342-1349, Antman et al. and pages 1333-1341, Ohman et al.
The Troponin complex is present in both skeletal and cardiac muscles and consists of three subunits, Troponin T ("TnT") the tropomyosin binding subunit, Troponin C ("TnC"), the Ca++ binding subunit and TnI, which inhibits the actomyosin Mg++-ATPase.
The majority of the research into the troponin complex has centered around the regulatory function and structure of the troponin complex in skeletal muscle. The troponin complex assists in muscle contraction. The TnC molecule has four binding domains to bind divalent metal ions. The Ca++/Mg++ binding sites are in the COOH terminal region and the Ca++ binding sites are in the amino terminal region. In studies of skeletal muscle, in the absence of Ca++, the amino terminus of TnI binds to the COOH terminus region of TnC and to the globular COOH terminus region of TnT. Thus, research indicates that TnI and TnC are anti-parallel and TnI and TnT are anti-parallel. The presence of calcium ion increases the TnC amino terminus domain's affinity for the inhibitory and COOH regions of TnI. In addition, there is a hydrophobic surface in the N-terminal domain of TnC that represents a Ca++ dependent binding site for TnI and TnT. It has been proposed that the Ca++ dependent reactions relate to the regulatory mechanism and Ca++ independent interactions maintain the structural integrity of the complex. In order to study structure and function of the troponin complex in its regulation of skeletal muscle, cross-linking studies have been accomplished. See, Farah, C. and Reinach, F. Review: The Troponin complex and regulation of muscle contraction. FASEB Journal 9 pp. 755-767 (1995). Covalent binding between TnC and skeletal muscle TnI has been formed between the carboxyl groups in the TnC and lysine groups in TnI using EDC. Kobayoshi et al. (1994), Structure of the troponin complex: implications of photocross-linking of troponin I to troponin C thiol mutants. J. Biol. Chem. 269, 5725-5729. In addition Leszyk et al. (1987) Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-malemidobenzophenone; identification of residues in troponin I that are close to cystein-98 of troponin C. Biochemistry 26, 7042-7047, reported that the main product of cross-linking between TnC and skeletal muscle TnI comprises segments derived from the N-terminal regulatory domain of TnC (residues 46 to 78) and the inhibitory region of skeletal TnI (residues 96-116). The Troponin complex is also referred to herein as the ternary complex.
U.S. Ser. No. 08/865,468, filed on May 29, 1997 and also owned by applicant, discloses that it had been discovered that the majority of native cTnI in human serum after an MI is associated with TnC and TnT. The presence of TnI in a complex with other troponin subunits in MI patient serum increases its stability and protects it from further degradation. In addition the troponin complex protects the sites where cardiac-specific antibodies bind. U.S. Ser. No. 08/865,468, filed on May 29, 1997 also discloses methods to isolate the complex from MI patient serum.
The cardiac isotype of the myofibrillar contractile protein, Troponin I ("TnI"), is uniquely located in cardiac muscle. TnI is the inhibitory sub-unit of Troponin, a thin filament regulatory protein complex, which confers calcium sensitivity to the cardiac and striated muscle. Troponin I exists in three isoforms: two skeletal TnI (fast and slow) isoforms (Molecular Weight=19,800 daltons) and a cardiac TnI ("cTnI") isoform with an additional 31 residues (human TnI) on the N-terminus resulting in a molecular weight of 23,000 daltons.
Cardiac TnI is found in human serum rapidly (within approximately 4 to 6 hours) following an MI. It reaches a peak level after approximately 18-24 hours and remains at elevated levels in the blood stream for up to 6 to 7 days. Thus, immunoassays which can test for human cTnI are valuable to the medical community and to the public.
It is desirable to use an immunologically reactive human cTnI isoform comparable to that found in MI patient serum. We found that MI patient serum contains TnI fragment(s) which is the result of the C-terminal processing of cTnI molecule. The high sequence homology found in the C-terminal region between cardiac TnI and skeletal muscle TnI (Larue et al. 1992 Molec. Immunology 29, 271-278, Vallins et al. 1990 FEBS Lett. 270, 57-61, Leszky et al. 1988 Biochemistry 27, 2821-2827) produce TnI antibodies directed against this region having non-cardiac specificity (Larue et al. 1992). Our data and Larue et al. 1992 suggest that most of the known cTnI specific antibodies have their epitopes located approximately in the first 75% of the TnI molecule. Therefore, this portion of the TnI molecule should function as a MI specific cTnI isoform in most immunoassay systems.
Currently cTnI immunoassays are commercially available from Dade International, Behring Diagnostics, and Sanofi Pasteur Diagnostics. The Dade product is the StratusO Cardiac Troponin-I assay.
Native intact human cTnI is difficult to obtain because of the scarcity of human heart and native intact human cTnI is highly subject to proteolytic degradation during purification. Recombinant cardiac TnI ("r-TnI"), unlike the native human cTnI, can be produced and purified in acceptable quantities. As expressed by Dade, the primary structure of r-TnI contains 226 amino acids (SEQ ID NO: 1); 209 of them represent the TnI sequence (SEQ ID NO: 2). In addition to the primary sequence of cTnI (SEQ ID NO: 2), r-TnI, as expressed by Dade International, has a leading sequence of 8 amino acids (MASMTLWM) on the N-terminal, and a tail sequence of 9 amino acids (PMVHHHHHH) on the C-terminal (SEQ ID NO: 1). The primary structure of the r-TnI molecule has methionine residues at positions -7, -4, 0, 153, 154, 200 and 211 (SEQ ID NO: 1). See also FIG. 1.
Full length cardiac troponin I is known to have the following sequence:
ADGSSDAAREPRPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQLKTLLLQIAKQELEREAEERRGEKGRAL STRCQPLELTGLGFAELQDLCRQLHARVDKVDEERYDIEAKVTKNITEIADLTQKIFDLRGKFKRPTLRRVRISA DAMMQALLGARAKESLDLRAHLKVKKEDTEKENREVGDWRKNIDALSGMEGRKKKFES (SEQ ID NO: 2) (Armour, K. L. et al.,(1993) Cloning and Expression in Escheria Coli of the cDNA Encoding Human Cardiac Troponin I, Gene, 131 (2):287-292).
U.S. Ser. No. 08/564,526, also owned by applicants, and incorporated herein by reference, discloses the use of a human cTnI fragment generated from human r-TnI by chemical cleavage. The cleavage of r-TnI by cyanogen bromide (CNBr) results in a major polypeptide of 153 amino acids, hereinafter referred to as the "ICNBr-cTnI isoform" (SEQ ID NO: 3). See FIG. 2. The CNBr-cTnI isoform represents 73% of the primary structure of human cTnI and is immunologically more reactive than r-TnI. The purified CNBr-cTnI isoform has an average of 3-4 times more reactivity than r-TnI and lower non-specific binding, as measured by radial partition immunoassay. The molecular size of the CNBr-cTnI isoform is comparable in molecular weight to the major degradation product of native cardiac TnI in MI patient serum.
It is desirable to use an immunologically reactive human cTnI isoform comparable to that detected in MI patient serum. The availability of r-TnI can facilitate the production of cardiac cTnI isoforms. Moreover, since most of the known human cardiac specific TnI antibodies have their epitopes located approximately in the first 75% of the TnI molecule, that portion of the TnI molecule will function as a cTnI isoform in most immunoassays.
The CNBr-cTnI isoform can be used as calibrators or controls in various cTnI immunoassays.
U.S. Ser. No. 98/865,468, filed May 29, 1997, discloses the use of cardiac troponin I fragments of the general sequence X-A-B-Y wherein X comprises any of amino acids 1-27 of full length cardiac troponin I, A comprises residues 28-69 of full length cardiac troponin I, B comprises amino acid residues 70-90 of full length cardiac troponin I, and Y comprises any sequential amino acid sequence of amino acid residues 91-170 of full length cardiac troponin I. These sequences also have increased immunoreactivity and stability over prior art compounds.
Troponin T (TnT) with a molecular weight of 39,000 Kd is part of the troponin-tropomyosin complex of the thin filament that is part of the muscle contractile apparatus and that contains actin and tropomyosin regulatory elements. Skeletal muscle studies of TnT have found that TnT is structurally asymmetric. Its terminal globular COOH terminal domain (TnT-2) mediates its interaction with TnI and TnC. TnT-1 at the amino terminus domain interacts with tropomyosin. See, Farah, C. and Reinach, F. (1995) Review: The Troponin complex and regulation of muscle contraction. FASEB Journal 9 755-767. It is reported that skeletal TnT is cleaved into the skeletal TnT-1 and TnT-2TnI-TnC fragments by mild proteolysis. Schaertl, S. et al. (1995) Separation and Characterization of the Two Functional Regions of Troponin Involved in Muscle Thin Filament Regulation. Biochemistry 34 (49) 15890-15894. TnT serves as a link between the tropomyosin backbone and the Troponin I/Troponin C complex. TnT has isotypes in cardiac and fast and slow skeletal muscles. It appears in serum about 3 hours after the onset of chest pain and remains elevated for at least 10 days following MI. Despite its lack of complete cardiac specificity it can be useful because of its rapid appearance into the bloodstream. Troponin T can be obtained as described in J. Biochem. 72: pages 723-735 (1972) or J. Biol. Chem. 249: 4742-4748, or purchased commercially. TnT gene promoter and derivatives thereof are disclosed in U.S. Pat. No. 5,266,488. TnT isoforms of skeletal muscle show variation in a given species in about a 30 amino acid region of the amino terminus and about a 14 amino acid region of the carboxy terminus. Pan, B. S. and Potter, J. D.(1992) Two Genetically Expressed Troponin T fragments Representing .alpha. and .beta. Isoforms Exhibit Functional Differences. Journal of Biological Chemistry 267 (82) 23052-23056.
In vitro stabilized solutions for cardiac markers have been disclosed. U.S. Pat. No. 5,583,200 and Bodor et al., (1992) Development of Monoclonal Antibodies for an Assay of Cardiac Troponin-I and Preliminary Results in Suspected Cases of Myocardial Infarction, Clinical Chemistry 38, (11) 2203-2214 at 2204 disclose stabilized troponin T and/or troponin I using troponin C and calcium ion. U.S. Pat. No. 5,583,200 discloses that serum may be added. U.S. Ser. No. 08/874,566, filed Jun. 13, 1997, discloses improvements in stabilizing the troponin T or troponin I/troponin C complex and discloses solutions useful as calibrators or controls for diagnostic assays measuring troponin. U.S. Ser. No. 08/564,526 and U.S. Ser. No. 08/865,468, filed May 29, 1997 also disclose the effect of TnC upon the immunological and biological activity and non-specific binding of the CNBr-cTnI isoform and other fragments. U.S. Ser. No. 08/564,526 discloses the activity of the complex formed by the CNBr-cTnI isoform, TnC and TnT as useful in immunoassays.
The calibrators and controls in Behring's OPUS.RTM. assay are a lyophilized preparation of human cardiac troponin I in processed bovine calf serum with stabilizers. The reconstituted products are stable for seven days when stored at 2 to 8 C. The calibrators and controls in Sanofi Pasteur's troponin I assay are a lyophilized preparation in a buffered human serum matrix. The reconstituted calibrators must be used within fifteen minutes after complete reconstitution, but may be aliquoted and stored frozed at -20 C for up to about six months. The calibrators and controls in the Dade troponin I assay are provided frozen. When thawed the product is stable for thirty days when stored at 2-8 C.
Improved methods and compositions of stabilized troponin I and/or troponin T are still necessary because under certain conditions the complex can dissociate (e.g. removal of calcium, presence of detergents such as SDS). Moreover, the closer the analyte used in the composition is to the actual circulating isoform of the troponin complex, the better the composition will perform as a primary reference material --that is a calibrator on which other calibrators are based.